The construction of immunogenic peptides or peptide conjugates is an active and ongoing research pursuit. The goals include production of reagent antibodies for research, for example in neurobiology, and production of synthetic vaccines for human or veterinary application. Small synthetic peptides are poor antigens and typically require covalent association with macromolecular carriers and administration with adjuvant in order to elicit an immune response. Carriers also provide T-cell epitopes necessary for cell-mediated response and for helper functions in the humoral response. When presented appropriately, synthetic peptides can elicit antibodies against large proteins which display the same peptide epitope within their sequence. Vaccine research further seeks to define those synthetic immunogens capable of inducing an antibody response that is also able to neutralize the infectious activity of a virus or other pathogen from which the protein is derived.
A significant effort has been devoted to discovery of general rules which govern the selection of protein epitopes by the immune system and development of methodology for mimicry of such epitopes with synthetic immunogens. Evidence has emerged suggesting that linear or discontinuous epitopes may be recognized and that these may adopt defined conformational states that are not readily duplicated in a synthetic peptide. Attempts to devise conformationally restricted peptides as superior antigens have also been given serious attention. In such approaches the structural context of a peptide sequence within a protein antigen is considered in producing a suitable mimic. Typically, the epitope may adopt a secondary structure such as a xcex2-turn or xcex1-helix. A similar structure may be induced in a small peptide by intramolecular covalent modification between two residues that constrains its conformational freedom. These ordered structures can be important as B-cell determinants. However, they are insufficient immunogens in the absence of helper T-cell determinants. Recently developed peptide vaccine models have incorporated T-cell epitopes in association with the B-cell epitope. Designs include simple tandem linear synthesis of peptides as well as increased epitope valency through coupling T-cell and B-cell peptides to a branched polylysine oligomer. The latter assemblies, referred to as multiple antigenic peptides, have shown promise as vaccines against various pathogens.
Unlike the conformationally defined B-cell epitopes, sequences recognized by T-cells undergo extensive processing to short linear peptide fragments before they are bound to a major histocompatibility complex (MHC) for recognition at the surface of an antigen-presenting cell. This elaborate processing mechanism depends on intracellular proteolytic activity and translocation of the products to the cell membrane. Synthetic peptide immunogens may not effectively participate in this process, despite the presence of the T-cell epitope. The immunogenicity of the molecule can be expected to correlate with the efficiency of natural processing of the T-cell epitope. Studies with linear synthetic peptides indicated that chimeric peptides containing T-cell and B-cell epitopes were superior immunogens when the B-cell epitope was amino-terminal. However, the reverse orientation has also been reported to produce a stronger immune response. A general rule may not be obvious since natural antigen processing probably accepts various orientations, including internal epitopes that require multiple processing steps to release the peptide. Also the efficiency of a construct may depend on many other factors, such as molecular context and flanking sequences that affect processing or presentation and the overall nature of the immunogen which can affect the functional pairing between several available T-cell and B-cell epitopes.
Further limitations to the use of synthetic peptides as vaccines result from the genetic restriction to T-cell helper function. Multiple MHC class II molecules encoded within the genome of a species are subject to allelic exclusion. A specific T-cell epitope may interact with only one or a few alleles of the MHC. Therefore individuals may respond differently to the immunogen despite inclusion of T-cell helper epitopes. Vaccine development must overcome the MHC-restricted response to provide the broadest possible response in an outbred population. In the murine model the T-helper cell responses are MHC-restricted and major haplotypes H-2b, H-2k, and H-2d are represented in several inbred strains. Certain T-cell epitopes are known to be recognized in the context of multiple MHC class II alleles and can thereby provide xe2x80x9cpromiscuousxe2x80x9d T-helper stimulation. A number of epitopes, such as from tetanus toxin, measles virus, and Mycobacterium tuberculosis have been reported to be universally immunogenic. These may have significant benefit for subunit vaccine design.
As an alternative to chemical synthesis, molecular biological techniques can provide significant advantages for production of polypeptides that display both B-cell and T-cell epitopes. Expression of proteins from cloned genes obviates burdensome peptide synthesis, purification and conjugation chemistry typically used in production of immunogenic materials. Furthermore, the stochastic chemistry for preparation of peptide-carrier conjugates is replaced by the defined chemical structure provided by the genetic fusion. Therefore the epitopes can be introduced in ordered structures that have optimal and reproducible immunogenic properties. Considerations that arise in the development of optimal designs can be addressed at the genetic level. Thus, definition of the target epitopes and their flanking sequences, relative orientation and conformations of these sequences within the larger polypeptide, and epitope copy frequency can be established in the gene design.
Several recombinant host proteins have been successfully utilized for immune presentation of peptide epitopes. The E. coli maltose-binding protein (MalE) has been used to study the influence of location and orientation of inserted T-cell epitopes. The major coat protein (pVIII) of filamentous bacteriophage fd has been used for display of HIV B-cell epitopes at the N-terminus. The recombinant phage particles evoked a strong antibody response in mice, which cross-reacted with HIV strains and which is also capable of neutralizing the virus. These approaches promise to enhance the potential of subunit or synthetic vaccine models.
The present invention relates to a variety of epitope-containing heat shock fusion proteins. In one embodiment, the heat shock protein ubiquitin is fused to a variety of eptiopes or epitope-containing segments. The specific fusion architecture is described in detail below. The epitope-containing segments of the ubiquitin fusion protein comprise either a single epitope or a group of identical or non-identical epitopes.
The present invention also relates to DNA constructs which encode an epitope-containing heat shock fusion protein of the type described above, and to cells transformed with such expression constructs.
In other aspects, the present invention relates to methods for stimulating an immune response in an animal, the immune response being directed toward a heat shock fusion protein of the type described above. The heat shock fusion protein is administered to an animal under conditions appropriate for the stimulation of an immune response. In an alternative embodiment, a DNA construct encoding the heat shock fusion protein is introduced into cells, rather than the fusion protein directly.
The present invention also relates to methods for inducing the production of antibodies to endogenous biomolecules using a heat shock fusion protein as described above where the said peptide epitopes are, or are not, related to the endogenous biomolecules in chemical composition but are so called xe2x80x9cmimicsxe2x80x9d of the biomolecular structure and as such have the ability to elicite antibodies to the said biomolecules. Such peptide (epitope) mimics are isolated typically by using phage peptide libraries.
The present invention also relates to methods for reducing levels of a predetermined protein (e.g., hormone(s)) in an animal relative to base-line levels, to methods for reducing endogenous TNF levels relative to those in a disease state, to methods for reducing the sperm count in males or inactivating sperm in men and women, to methods for reducing the allergic response, to methods for increasing the growth rate of an animal and to methods for the production and identification of antibodies for use in experimental or diagnostic samples.
The present invention is based, in one aspect, on the discovery that a fusion protein comprising a heat shock protein (e.g., ubiquitin), fused to an epitope or epitopes in a defined manner, is useful for the stimulation of a highly specific immune response when administered to an animal. The specific fusion architecture encompassed by the invention will be discussed in greater detail below.
Heat shock proteins are proteins which are induced during a heat shock. Other stress stimuli are also known to induce a similar response. These proteins are produced to enable the cell to better stand the heat shock or stress. Under these conditions the pattern of gene expression changes and cells overproduce a characteristic set of proteins commonly referred to as heat-shock proteins (hsps). One factor in the induction of the heat shock response is the proteolysis of abnormal proteins.
Various examples of heat shock proteins exist. In yeast, mammals and other eukaryotes, ubiquitin is one of these proteins. Ubiquitin is known to be involved in an ATP-dependent pathway of proteolysis. It is also known that proteolysis is an important step in the production of peptides which function in development of the immune response to antigens. Thus it is recognized that hsps are ideal candidates for use in connection with vaccine development.
In connection with the present invention, ubiquitin is used as a scaffold to stabilize and display recombinant immunologically active heterologous antigens (referred to herein as epitopes), presumably in a form which generally approximates the native conformation of the epitope. This technique is sometimes referred to as conformational mimicry. Generally, the preferred size of the amino acid segments referred to in items a) and b) of the preceding paragraph range from about 5 to about 70 amino acid residues, although larger segments are not intended to be excluded by this statement of the preferred size range.
Ubiquitin is a small 76-residue single domain protein which does not induce an appreciable immune response when administered to an animal. Presumably, this xe2x80x9cimmunological silencexe2x80x9d is based on the fact that ubiquitin is expressed in nearly identical form in all eukaryotic systems. Ubiquitin has a variety of other characteristics which make it an ideal xe2x80x9ccarrierxe2x80x9d for the conformational mimicry approach. For example, ubiquitin is highly resistant to protease digestion and is extremely stable both in vivo and when stored for extended periods of time in vitro. The three-dimensional structure of ubiquitin has been determined by X-ray crystallography and its small size makes it amenable to molecular modeling. Additionally, ubiquitin fusions can be overexpressed in prokaryotic systems such as E. coli, in soluble form, and purified. One of skill in the art will recognize that for particular applications some of the aforementioned properties are unimportant and dispensable. At the present time, there is no comparable protein scaffold system available which offer the benefits of the ubiquitin system.
If epitopes are to be fused to the ubiquitin framework as outlined above, whether at a single location or non-contiguous locations, it is important to determine what types of ubiquitin modifications are tolerated. In this context, xe2x80x9ctoleratedxe2x80x9d can have at least two meanings, systemic tolerance and functional tolerance. It should also be noted that the expression xe2x80x9cfusedxe2x80x9d, as used herein, means covalent bound by an amide linkage. This expression encompasses insertion, as well as substitution. At times, these expressions may be used interchangeably herein.
Systemic tolerance, (i.e., tolerance by the immune system) is important to ensure that the immune response is directed to the epitopes, and not to the ubiquitin carrier. Thus, epitope insertion should be designed such that changes to the secondary and tertiary structure of ubiquitin are minimized.
Ubiquitin functional tolerance refers to the ability of the ubiquitin protein to behave functionally in a manner analogous to wild-type ubiquitin. This functional tolerance can also be important in a variety of contexts. This property should also be maintained, at least in connection with certain applications, by the ubiquitin fusion carrier constructs of the present invention.
As disclosed herein, insertions at particular internal sites in ubiquitin are xe2x80x9ctoleratedxe2x80x9d, as this term is defined above. One advantage of using ubiquitin as an immunogenic display scaffold, particularly for an internally-fused epitope or epitopes, resides in its ability to maintain the secondary structure found in the epitopes native protein. These secondary structures include, for example, xcex2-turns and xcex1-helices. The conformation of the epitope can be further modified by introducing intramolecular bonds between two residues of the epitope which results in conformational constraints on the overall structure of the epitope. The fusion of an epitope or epitopes to terminal regions of ubiquitin also offers advantages in connection with immune-stimulatory activities.
In a first embodiment, the present invention relates to a ubiquitin fusion protein comprising ubiquitin fused to a single epitope-containing segment comprising two or more identical or non-identical epitopes, the epitope-containing segments being fused to ubiquitin at fusion sites selected from the group consisting of the N-terminus and an internal fusion site. As discussed in greater detail below, a variety of considerations are taken into account when selecting an epitope of use in connection with the fusion proteins of the present invention. Generally speaking, an epitope which can stimulate an immune response which protects against an infectious disease, an auto-immune disease or allergic reactions are candidate epitopes for use in connection with the present invention. Epitopes which do not fall into one of these categories can also be useful, and non-limiting examples are discussed more fully below.
With respect to the first embodiment, fusions at a single internal location in the ubiquitin moiety must be designed rationally to minimize, for example, adverse consequences with respect to ubiquitin structure and function. In light of the fact that fused epitopes must be xe2x80x9cseenxe2x80x9d by the immune surveillance system, it is also important that internally fused epitopes are exposed in the folded fusion protein, not buried within a hydrophobic domain. The plurality of epitopes can also be fused to ubiquitin at the N-terminus of the molecule. The epitopes can be identical or non-identical. In addition, the epitopes can be B cell epitopes, T cell epitopes or a mixture of B and T cell epitopes. For many applications, preferred epitopes are B-cell epitopes which are known to be a target for neutralizing antibodies.
A second embodiment of the present invention relates to a ubiquitin fusion protein comprising ubiquitin fused to two or more non-contiguous epitope-containing segments, each epitope-containing segment comprising one or more identical or non-identical epitopes. The non-contiguous locations where fusion is appropriate are internal locations within the ubiquitin moiety, or at the N- or C-terminus of the ubiquitin molecule.
As used herein in connection with the second embodiment, the term xe2x80x9cepitope-containing segmentxe2x80x9d refers to a sequence of amino acids containing one or more epitopes. The epitopes within any particular epitope-containing segment can be identical, or non-identical. In addition, the epitopes in a particular epitope-containing segment can be B cell epitopes, T cell epitopes or a mixture of B and T cell epitopes. As discussed above, B-cell epitopes targeted by neutralizing antibodies are preferred in some contexts.
When considering the insertion of epitopes within the ubiquitin molecule, the structure of the ubiquitin molecule must be considered. The prominent structural features of ubiquitin, as determined by X-ray crystallography (see, e.g., Vijay-Kumar et al., Proc. Natl. Acad. Sci. USA 82: 3582 (1985); Vijay-Kumar et al., J. Mol. Biol. 194: 525 (1987); and Vijay-Kumar et al., J. Biol. Chem. 262: 6396 (1987)) include a mixed xcex2-sheet comprising two parallel inner strands (residues 1-7 and 64-72), as well as two antiparallel strands (residues 10-17 and 40-45). In addition, an xcex1-helix (residues 23-34) fits within the concavity formed by the mixed xcex2-sheet.
The amino acid sequences which link the structural elements defined in the preceding paragraph are referred to herein as xe2x80x9cloop regionsxe2x80x9d. Thus, loop regions can be defined as domains of ubiquitin which link either two strands within a xcex2-sheet or a strand of a xcex2-sheet and an xcex1-helix. Insertions and substitutions can be made within these loop regions without disrupting the integrity of the ubiquitin molecule or abolishing the features which make ubiquitin a useful carrier for the display of constrained epitopes. Insertions and substitutions within these loop regions tend not to alter the relationships between the prominent structural features defined in the preceding paragraph. Rather, the epitopes introduced into these loop regions tend to protrude from the compact globular ubiquitin structure thereby exposing these epitope residues such that they are easily recognizable by lymphocytes, for example.
As discussed above, internal modification sites are selected such that the ubiquitin secondary structure is maintained and the conformation of the inserted epitope is constrained. Epitopes can also be joined to ubiquitin as extensions of the C-terminus. Epitopes fused to the C-terminus of ubiquitin can be cleaved off by ubiquitin-specific proteases in vivo or in vitro. This allows the peptide to be administered to a cell as part of a larger fusion protein which is both easier to purify and handle as compared to free epitope. Following cellular uptake, the epitope attached to the ubiquitin can be cleaved from the C-terminus of ubiquitin and associated with a surface protein such as the MHC complex for expression on the cellular membrane.
In another embodiment, the subject invention relates to a ubiquitin fusion protein comprising ubiquitin fused to a single epitope-containing segment, the epitope-containing segment comprising two or more identical or non-identical epitopes. The epitope-containing segment can be fused to ubiquitin at its N-terminus, terminus or internally.
The invention relates to yet another fusion protein embodiment comprising ubiquitin fused to a single epitope-containing segment comprising one or more identical or non-identical epitopes. In this embodiment, the epitope-containing segment is fused to the ubiquitin moiety at the N-terminus of ubiquitin.
The use of ubiquitin fusion proteins to initiate a humoral response is described in more detail in the following Exemplification section. More, specifically, these experiments demonstrate, for example, that the B-cell and T-cell epitopes expressed in the ubiquitin fusion protein stimulated targeted immune responses. Further, the experiments demonstrate that a humoral immune response to an internally inserted B-cell epitope was enhanced by the addition of a T-cell epitope to the C-terminus of the ubiquitin fusion protein. Although the bulk of the in vivo data reported herein were generated in experiments employing murine indicator assays for the generation of antibodies against the ubiquitin fusion proteins, the fundamental principles are applicable to humans as well as other animals. Given the disclosure of the subject application it is a matter of routine experimentation to select epitopes of interest and incorporate such epitopes of interest into a ubiquitin fusion protein for use as an immunogen.
Thus, in a preferred embodiment, the ubiquitin fusion protein comprises an internally inserted B-cell epitope and a T-cell epitope joined to the C-terminus of ubiquitin. One of skill in the art can identify B-cell epitopes which have the ability to drive a strong humoral immune response following administration to an animal. The B-cell epitope which is selected will depend upon the intended use of the ubiquitin fusion protein. For instance, if the ubiquitin fusion protein is to be used as a vaccine, the B-cell epitope can be derived from a protein which is expressed by a virus, bacteria or other infectious organism associated with causing a disease. The protein which is selected should be one which contains epitopes which elicit strong antibody responses. These responses are associated with protection of the animal species from the symptomology caused by the infectious organism. Preferably, the B-cell epitope which is selected is derived from a portion of the protein from the infectious organism known to be both highly immunogenic and to which protective antibodies can be produced. In general, this will include proteins found on the surface of the infectious organism which are involved in binding and to which antibodies have a high degree of access.
One example of a B-cell epitope which fulfills the requirements set forth above is the V3 loop of the HIV gp120 glycoprotein. As described in the following section, an epitope derived from the V3 loop of the HIV gp120 glycoprotein, when internally inserted within a ubiquitin fusion protein, is able to drive a strong humoral response. In fact, the antibody response was stronger than that found when the epitope was administered independently as a peptide antigen. The selection of this epitope was based on extensive data showing it is a target of neutralizing antibodies.
The selection of the B-cell epitope is not limited to proteins associated with infectious organisms. For instance, as shown in Example 2 below, when the ubiquitin fusion protein contains an internally inserted epitope from a prostate-specific antigen, a strong antibody recognition was detected. Examples of other epitopes useful in connection with the present invention include those from proteins which are commonly used as immunological xe2x80x9ccarriersxe2x80x9d as part of experimental studies. These include hen egg lysozyme, keyhole limpet hemocyanin and ovalbumin. One of skill in the art will recognize that any protein containing a B-cell epitope which is capable of driving a humoral immune response can be included in a fusion protein of the present invention. Many such epitopes are known and others can be determined through routine experimentation.
In a preferred embodiment, a T-cell epitope is joined to the C-terminus of the ubiquitin fusion protein. This epitope is selected based on its ability to enhance the humoral immune response directed to the internally inserted B-cell epitope when both are placed in their proper orientation with respect to ubiquitin. The preferred T-cell epitope is selected from a group of T-cell epitopes which are able to elicit xe2x80x9cpromiscuousxe2x80x9d T-cell help. This type of T-cell epitope is commonly referred to as a universal epitope. Universal T-cell epitopes function by stimulating helper T-cells specific to the B-cells responsive to the ubiquitin fusion protein containing the internally inserted B-cell epitope. They do this regardless of the subject""s MHC haplotype or whether the specific xe2x80x9ctargetxe2x80x9d protein is different than the protein the universal T-cell epitope is derived from. Examples of universal T-cell epitopes include epitopes from tetanus toxin, measles virus and Mycobacterium tuberculosis. This list of universal T-cell epitopes is not intended to be comprehensive, others which are known or can be determined through application of routine experimentation are also included.
To stimulate cytotoxic T-cells as part of a cellular immune response, T-cell epitopes are preferably inserted internally within the ubiquitin moiety. In addition, it is preferable to fuse at least one T-cell epitope to the C-terminus of the ubiquitin fusion protein. In this case, the T-cell epitopes are selected on the basis of their ability to ensure stimulation of cytotoxic T-cells specific for the particular epitope. However, a universal T-cell epitope can be attached to the C-terminus of the ubiquitin fusion protein to enhance the stimulation of the specific T-cell response.
Cytotoxic T-cells play an important role in the surveillance and control of viral infections, bacterial infections, parasitic infections and cancer, for example. Vaccination with synthetic epitopes, or with epitope pulsed cells, has been shown to induce specific cytotoxic T-cell responses directed against immunogenic viral or tumor-derived epitopes. Alternative protocols of T-cell activation allow the triggering of more selective cytotoxic T-cell responses with greater therapeutic effectiveness.
Generally, the fusion of peptides to the C-terminus of ubiquitin generates a construct which is cleavable, in vivo, by ubiquitin-specific proteases. It is well-established that such ubiquitin-specific proteases cleave ubiquitin fusions after a C-terminal residue (residue 76), thereby releasing the C-terminal peptide. The present invention also encompasses ubiquitin fusion proteins which have been modified such that the fusion is not efficiently cleaved by ubiquitin-specific proteases. As is well known to those of skill in the art, ubiquitin can be made resistant to ubiquitin-specific proteases by altering residues at the C-terminus of ubiquitin. For example, by altering the identity of the amino acid at position 76 of ubiquitin (e.g., from glycine to valine or cysteine), the rate of cleavage of a C-terminal ubiquitin fusion can be substantially reduced to the point where cleavage can not be detected using the assays typically employed for monitoring such cleavage.
As mentioned previously, the present invention also encompasses fusions to the N-terminus of ubiquitin. It is noted that fusion proteins in which an epitope or epitopes are attached to the N-terminus ubiquitin must be designed such that the N-terminal residue of the encoded fusion protein is methionine. If the N-terminal residue is a residue other than methionine, initiation of translation does not occur efficiently. Unfortunately, however, previous studies have demonstrated that fusions of this type can reduce antibody binding or elicit the production of antibodies of low affinity for the epitope fused to the N-terminus of ubiquitin.
To overcome this problem, a tandem ubiquitin fusion is created by attaching a second ubiquitin protein to the N-terminus of the epitope or epitopes attached to the N-terminus of the first ubiquitin moiety. Thus, in this embodiment of the present invention, two ubiquitin molecules flank an epitope or epitopes. The C-terminus of the N terminal ubiquitin protein is of the wild-type sequence such that it is cleavable by ubiquitin specific proteases. DNA encoding this tandem ubiquitin fusion is used to transform either prokaryotic or eukaryotic cells. Previous work has shown that the tandem ubiquitin fusion is produced at an equivalent or increased level as compared to the single ubiquitin fusion proteins.
Following purification, the tandem ubiquitin fusion can be cleaved, in vitro, by a ubiquitin specific protease thereby releasing the N terminal ubiquitin protein from the core fusion protein which is comprised of an epitope attached to the N-terminus of the C terminal ubiquitin protein.
The C terminal ubiquitin protein in the tandem ubiquitin fusion protein can be modified by the inclusion of other epitopes in a manner consistent with the description of the invention above. Thus, fusion can be made within the C terminal ubiquitin molecule or to the C-terminus of the C terminal ubiquitin molecule.
An alternative tandem ubiquitin construct suitable for use in connection with all embodiments of the present invention is encompassed within the scope of the present invention. More specifically, in the alternative tandem ubiquitin construct, the two ubiquitin moieties are contiguous (i.e., the N-terminus of a first ubiquitin moiety is joined to the C-terminus of a second ubiquitin moiety). In this embodiment, the N-terminal residue of the first ubiquitin moiety is a residue other than methionine. This first ubiquitin moiety is then fused to a wild-type ubiquitin moiety which is cleavable by a ubiquitin-specific protease.
Alternatively, the second ubiquitin moiety in this tandem construct need not be a complete ubiquitin moiety. Rather, a C-terminal subdomain of ubiquitin competent to direct cleavage by a ubiquitin-specific protease, is sufficient. In addition, in connection with this and any other embodiment of the present invention, ubiquitin (or portions thereof) may be modified to inhibit cleavage by a ubiquitin-specific protease.
A variant of one of the two major embodiments of the present invention is a ubiquitin fusion comprising a first and a second epitope-containing segment inserted internally, and a third epitope-containing segment fused to the C-terminus of ubiquitin. The subject invention encompasses a wide range of such variant embodiments. There is no theoretical limit on the number of epitopes which can be inserted within or fused to the N and C-terminus of a ubiquitin fusion protein.
In preferred embodiments, internally fused epitopes are fused as single epitopes, non-contiguously. This design ensures that antibodies produced following vaccination are specific for a single epitope and do not cross-react with other epitopes which have also been internally fused to ubiquitin. Thus, each epitope elicits a specific antibody response by producing antibodies which do not cross-react with other epitopes contained within the same ubiquitin fusion protein. The use of an epitope-containing segment in which two or more distinct epitopes are displayed is preferred when attempting to create bifunctional antibodies for experimental, diagnostic or therapeutic uses.
In another embodiment of the present invention, an epitope-containing ubiquitin fusion protein is modified by conjugation to a carrier protein such as ovalbumin (OVA) or keyhole limpet hemocyanin (KLH). Example 5, presented below, exemplifies such an embodiment.
Ubiquitin fusion proteins of the type described above can be modified post-translationally by the addition of fatty acids to enhance immunogenicity. For example, palmatic acid (C16) can be added using appropriate chemistry for this purpose.
The discussion above has focused on a wide variety of epitope-containing ubiquitin fusion proteins. The invention also relates to DNA expression constructs which encode such epitope-containing ubiquitin fusion proteins. These constructs can be based on prokaryotic expression vectors or eukaryotic expression vectors. Many examples of such expression vectors are known in the art. Prokaryotic expression vectors are useful, for example, for the preparation of large quantities (e.g., up to milligram quantities) of the ubiquitin fusion protein. Eukaryotic expression vectors are useful, for example, when the addition of carbohydrate side chains (i.e., glycosylation) is important. The carbohydrate side chains can affect the properties of a protein in a variety of ways including, for example, the ability of the protein to function in vivo or in vitro; the ability of the protein to form a complex and associate with other proteins or nucleic acids; and the ability of the protein to bind to an antibody or other molecules specific for the protein of interest.
In another aspect, the present invention relates to methods of vaccination. The vaccine can be used to drive a cellular and/or humoral immune response depending on the type of epitopes fused to the ubiquitin fusion protein. The therapeutic amount of the ubiquitin fusion protein given to an animal species will be determined as that amount deemed effective in eliciting the desired immune response. The ubiquitin fusion protein is administered in a pharmaceutically acceptable or compatible carrier or adjuvant.
Thus, the present invention also encompasses pharmaceutical compositions for the administration of ubiquitin fusion proteins. Examples of specific diseases which can be treated in this manner include, for example, gastrointestinal diseases, pulmonary infections, respiratory infections and infection with HIV. The pharmaceutical compositions are prepared by methods known to one of skill in the art. In general, the ubiquitin fusion protein is admixed with a carrier and other necessary diluents which are known in the art to aid in producing a product which is stable and administrable. Administration of the pharmaceutical composition can be accomplished by several means known to those of skill in the art. These include oral, intradermal, subcutaneous, intranasal, intravenous or intramuscular.
Conventional vaccination methods involve the administration of an epitope-containing protein. Recently, and alternative to conventional vaccination methods, referred to as DNA vaccination, has been developed. In this method, DNA encoding the epitope-containing protein is introduced into the cells of an organism. Within these cells, the epitope-containing protein is directly expressed. Direct expression of the ubiquitin fusion proteins of the present invention by endogenous cells of a vaccinated animal allows for the continual stimulation of humoral and cellular immune responses over an extended period of time. This is in contrast to standard immunization protocols whereby the vaccine is injected at a single site one or more times. Following injection, the vaccine is disseminated to lymphoid organs where a single immune response occurs.
Direct expression can be accomplished by introducing DNA constructs which encode the desired ubiquitin fusion protein into the cells of an animal. The constructs typically contain promoter elements and other transcriptional control elements which direct the expression of the ubiquitin fusion protein. Introduction of the DNA construct can be by any conventional means including direct injection. The preferred administration site is muscle tissue or tissues rich in antigen presenting cells.
The introduction of a ubiquitin fusion protein as described above can also induce a tolerizing effect on the humoral or cellular immune response in an animal. Tolerization occurs following delivery of the ubiquitin fusion proteins to T-cells. The induction of a tolerization response is useful, for example, in connection with the treatment of allergic or autoimmune disorders. Examples of epitopes which can be used in a therapeutic regimen designed to induce tolerization include the Fel d 1 peptides, which are the major allergens found in cat pelts. These peptides can be internally inserted, for example, and fused to a cleavable C-terminus of ubiquitin. Typically patients to be treated are dosed subcutaneously with the ubiquitin fusion proteins once per week for several weeks. However, dosing can also be done orally or intranasally over a similar length of time. The result is a reduction of the allergic and/or autoimmune responses. These ubiquitin fusion proteins can also be given orally.
The use of ubiquitin as a scaffold for the presentation and stimulation of immune responses also allows the stimulation and generation of anti-self responses. An example of a potentially valuable anti-self response is the generation of anti-GnRH (gonadotrophin releasing hormone) antibodies. As described in Example 3 below, efforts have been made to generate immunogens which stimulate a strong anti-GnRH response which results in the suppression of luteinizing hormone (LH) and follicle stimulating hormone (FSH) and indirectly suppresses the production of the steroidogenesis and gamete maturation in both males and females. The value of this type of anti-self response in humans lies in the treatment of prostate cancer and breast cancer.
In livestock and pets, the ability to stimulate an anti-self response provides a simple alternative to physical castration. Previous work employing complex immunogens has demonstrated varying degrees of success in immunological castration. However, the production of complex immunogens is cumbersome, typically involving a combination of synthetic chemistry, expensive HPLC purifications, and chemical coupling methods. The use of ubiquitin fusion proteins containing GnRH epitopes facilitates the production of inexpensive and potent immunogens for use in connection with immunocastration. In particular, ubiquitin fusions which include the peptide
QHWSYGLRPGQHWSYGLRPGQHWSYGLRPGQHWSYGLRPGC (SEQ ID NO: 34)
are of interest.
Immunocastration of pigs is especially valuable since it does not result in the detrimental side-effects associated with physical castration. More specifically, physical castration of pigs typically results in animals which do not grow as well as normal animals. In addition, physically castrated pigs tend to have a higher fat percentage than non-castrated pigs. Since, immunocastrated animals are not castrated as early as those which undergo normal physical castration, a farmer can take advantage of the growth rates found with non-castrated animals. Finally, by immunocastrating, the farmer avoids the production of unpalatable meat found with uncastrated male pigs.
Other examples of self proteins which could be used with ubiquitin to generate vaccines able to modulate the hormones, cytokines and physiology of humans and animals are: growth hormone and its peptides to modulate growth both negatively and positively; TNF and its epitopes to modulate septic shock, arthritis, inflammatory bowel disease, crohnys disease, and ulcerative colitis; immunoglobulin epsilon heavy chain for the control of allergic reactions; chorionic gonadotrophin for fertility control; inhibit for fertility control; and Sperm proteins such as sp17 (for example amino acids 4-19 and 118-127) and the 71 kd sperm protein for control of fertility both in men and in women.
A further use of ubiquitin as a scaffold is as part of a vaccine to enhance the growth rate and thereby the final weight of livestock prior to shipment to market. This type of vaccine offers a cost effective means to increase the value of livestock such as pigs, cattle and other commonly raised animals. Presently, to increase the weight of livestock several methods are being utilized. Amongst these methods, the addition of antibiotics to the feed has become very common. However, while addition of antibiotics to feed is cost effective, it has limitations. For instance, it has been blamed for an increase in the creation of antibiotic resistant strains of bacteria.
An alternative means to increase the growth rate of livestock which does not result in detrimental side-effects is through vaccination of the animals with an epitope of growth hormone which is part of a ubiquitin fusion protein. The result of this vaccination is an increase in the activity of the animals endogenous growth hormone. The vaccine is created by inserting an epitope from the growth hormone protein (e.g., amino acids 54-95) into the ubiquitin protein thus creating a ubiquitin-growth hormone fusion protein. The effective use of this type of vaccine is described in Example 7 below. More specifically, following the injection of a vaccine comprised of adjuvant and a ubiquitin fusion protein containing the growth hormone protein epitope, the growth rate of pigs was improved when compared to control pigs which received either adjuvant only or adjuvant and ubiquitin only.
In addition to the uses described above, the ubiquitin fusion proteins of the present invention can be used for the identification of antibodies from experimental or clinical samples. Antibodies to be assayed can be found, for example, in blood, fecal material, the linings of mucosal associated lymphoid tissues, cellular biopsies and other sources known by one of skill in the art to contain at least a minute quantity of antibody. Assays for which the ubiquitin fusion proteins are well suited include ELISAs, radioimmunoassay as well as other commonly used competition assays. These types of assays are useful in identifying antibodies from an experimental or clinical sample which have specificity to at least one epitope of a known protein. In general, the assays involve mixing a predetermined aliquot of the ubiquitin fusion protein with a series of dilutions of the experimental or clinical antibody sample. This is followed by detection of the antibodies which bind to the ubiquitin fusion protein.
Detection can be accomplished by various means, but for the present invention, a labeled detection antibody is preferable. For example, if the experimental or clinical antibody sample is from a human, the detection antibody can be a polyclonal antibody with specificity for the human heavy chain portion of the sample antibody. The detection antibody is attached to either an enzymatic label, a radioactive label or a fluorochromatic label. Examples of commonly used enzymatic labels are horse radish peroxidase and alkaline phosphatase. A standard radioactive label includes iodine 131. Fluorochromatic labels include fluorescein and Texas red. The method of visualization of the complex containing the ubiquitin fusion protein, the sample antibody, and the labeled antibody depends on the label attached to the antibody and would be known to those of skill in the art.